The present invention relates to the field of bioconjugate preparation, and more particularly, to a novel class of 1,2-phenylenediboronic acid (1,2-PDBA) reagents useful for the conjugation of biological macromolecules, and the method of making and using such reagents.
Bioconjugation is a descriptive term for the joining of two or more different molecular species by chemical or biological means, in which at least one of the molecular species is a biological macromolecule. This includes, but is not limited to, conjugation of proteins, peptides, polysaccharides, hormones, nucleic acids, liposomes and cells, with each other or with any other molecular species that add useful properties, including, but not limited to, drugs, radionuclides, toxins, haptens, inhibitors, chromophores, fluorophores, ligands, etc. Immobilization of biological macromolecules is also considered a special case of bioconjugation in which the macromolecule is conjugated, either reversibly or irreversibly, to an insoluble support including a chromatographic support. Bioconjugation is utilized extensively in biochemical, immuno-chemical and molecular biological research. Major applications of bioconjugation include, but are not limited to, detection of gene probes, enzyme-linked immunological solid-phase assay, monoclonal antibody drug targeting and medical imaging.
Bioconjugates are generally classified as either direct or indirect conjugates. Direct conjugates encompass those in which two or more components are joined by direct covalent chemical linkages. Alternatively, indirect conjugates encompass those in which two or more components are joined via an intermediary complex involving a biological macromolecule.
Although numerous methods of indirect bioconjugate preparation have been described, a significant number of those reported in the literature have been prepared by exploiting the Avidin-Biotin system. In the Avidin-Biotin system, the binding specificity of the protein Avidin (purified from egg white), or Streptavidin (purified from the bacterium Streptomyces avidinii), toward the cofactor Biotin (vitamin H) is utilized to bridge an Avidin conjugated macromolecule with a biotinylated macromolecule. Both Avidin and Streptavidin possess four Biotin binding sites of very high affinity (Kd=10xe2x88x9215 molxe2x88x921).
The Avidin-Biotin system has been utilized extensively for enzyme-linked immuno-logical solid-phase assay (ELISA), in which an enzyme-Avidin conjugate (useful for detection by reaction with the enzyme""s substrate to afford a colored or chemiluminescent product) is employed to detect the presence of a biotinylated antibody, after first binding the antibody to an immobilized antigen or hapten. Applications of the Avidin-Biotin system number in the hundreds, and have recently been reviewed (Wilchek, M. and Bayer, E. A., (1990) Methods in Enzymology, 184).
Although utilized extensively, several limitations are known to be associated with the Avidin-Biotin system, which include nonspecific binding generally attributed to the basicity of the Avidin molecule, nonspecific binding attributed to the presence of carbohydrate residues on the Avidin molecule, and background interference associated with the presence of endogenous Biotin, which is ubiquitous in both eukaryotic and prokaryotic cells.
An alternative indirect bioconjugation system designed to overcome some of the limitations associated with the Avidin-Biotin system has recently been developed for the detection of gene probes by ELISA. See Kessler, C., Hxc3x4ltke, H.-J., Seibl, R., Burg, J. and Mxc3xchlegger, K., Biol. Chem. Hoppe-Seyler (1990), 371, 917-965. This system involves the use of the steroid hapten Digoxigenin, an alkaloid occurring exclusively in Digitalis plants, and Fab fragments derived from polyclonal sheep antibodies against Digoxigenin (anti-Digoxigenin). The high specificity of the various anti-Digoxigenin antibodies affords low backgrounds and eliminates the non-specific binding observed in Avidin-Biotin systems. Digoxigenin-labeled DNA and RNA probes can detect single-copy sequences in human genomic Southern blots. The development of the Digoxigenin anti-Digoxigenin system has recently been reviewed. See Kessler, C. (1990) in Advances in Mutagenesis Research (Obe, G. ed.) pp. 105-152, Springer-Verlag, Berlin and Heidelberg. The Digoxigenin anti-Digoxigenin system is the most recent representative of several hapten-antibody systems now routinely utilized for bioconjugation.
Phenylboronic acids are known to interact with a wide range of polar molecules having certain requisite functionalities. Complexes of varying stability, involving 1,2-diols, 1,3-diols, 1,2-hydroxy acids, 1,3-hydroxy acids, 1,2-hydroxylamines, 1,3-hydroxylamines, 1,2-diketones and 1,3-diketones, are known to form with either neutral phenylboronic acid or the phenylboronate anion. Consequently, immobilized phenylboronic acids have been exploited as chromatographic supports to selectively retain, from diverse biological samples, those molecular species having the requisite functionalities. Many important biological molecules including carbohydrates, catecholamines, prostaglandins, ribonucleosides, and steroids contain the requisite functionalities, and have been either analyzed or purified in this manner. The use of phenylboronic acid chromatographic media for the isolation and separation of biological molecules has been discussed in several reviews. See Singhal, R. P. and DeSilva, S. S. M., Adv. Chromatog. (1989), 31, 293-335; Mazzeo, J. R. and Krull, I. S., BioChromatog. (1989), 4, 124-130; and Bergold, A. and Scouten, W. H. (1983) in Solid Phase Biochemistry (Scouten, W. H. ed.) pp. 149-187, John Wiley and Sons, New York.
Phenylboronic acid, like boric acid, is a Lewis acid, and ionizes not by direct deprotonation, but by hydration to give the tetrahedral phenylboronate anion (pKa=8.86). Phenylboronic acid is three times as strong an acid as boric acid. Ionization of phenylboronic acid is an important factor in complex formation, in that, upon ionization, boron changes from trigonal coordination (having average bond angles of 120xc2x0 and average bond lengths of 1.37 angstroms) to the tetrahedral coordination (having average bond angles of 109xc2x0 and average bond lengths of 1.48 angstroms).
Molecular species having cis or coaxial 1,2-diol and 1,3-diol functionalities, and particularly carbohydrates, are known to complex with immobilized phenylboronate anion, to form cyclic esters under alkaline aqueous conditions. See Lorand, J. P. and Edwards, J. O., J. Org. Chem. (1959), 24, 769.
Acidification of 1,2-diol and 1,3-diol complexes to neutral pH is know to release the diol containing species, presumably due to hydrolysis of the cyclic ester. Coplanar aromatic 1,3-diols, like 1,8-dihydroxynaphthalene, are known to complex even under acidic conditions due to the hydrolytic stability of six-membered cyclic boronic acid esters. See Sienkiewicz, P. A. and Roberts, D. C., J. Inorg. Nucl. Chem. (1980), 42, 1559-1571.
Molecular species having pendant 1,2-hydroxylamine, 1,3-hydroxylamine, 1,2-hydroxy-amide, 1,3-hydroxyamide, 1,2-hydroxyoxime and 1,3-hydroxyoxime functionalities are also known to reversibly complex with phenylboronic acid under alkaline aqueous conditions similar to those associated with the retention of diol containing species. See Tanner, D. W. and Bruice, T. C., J. Amer. Chem. Soc. (1967), 89, 6954.
The most popular methods of synthesizing phenylboronic acids involve in situ generation of arylmagnesium or aryllithium species from aryl halides followed by transmetalation with a trialkoxyborate. See Todd, M. H., Balasubramanian, S. and Abell, C., Tetrahedron Lett. (1997), 38, 6781-6784; Thompson, W. and Gaudino, J., J. Org. Chem. (1984), 49, 5237-5243; Crisofoli, W. A. and Keay, B. A., Tetrahedron Lett. (1991), 32, 5881-5884; Sharp, M. J., Cheng, W. and Sniekus, V., Tetrahedron Lett. (1987), 28, 5093-5096; and Larson, R. D., King, A. O., Cheng, C. Y., Corley, E. G., Foster, B. S., Roberts, F. E., Yang, C., Lieberman, D. R., Reamer, R. A., Tschaen, D. M., Verhoeven, T. R. and Reider, P. J., J. Org. Chem. (1994), 59, 6391-6394.
Recently, transition-metal catalyzed cross coupling reactions have been developed to produce phenylboronic acids from aryl halides and alkoxydiboron, see Ishiyama, T., Murata, M. and Miyaura, N., J. Org. Chem. (1995), 60, 7508-7510; Giroux, A., Han, Y. and Prasit, P., Tetrahedran Lett. (1997), 38, 3841-3844, or dialkoxyhydroborane, see Murata, M.; Watanabe, S.; Masuda, Y., J. Org. Chem. (1997), 62, 6458-6459 using PdCl2 (dppf) as the catalyst. Additionally, a palladium-catalyzed solid-phase boronation, using alkoxydiboron, has also been reported using a polymer-bound aryl halide. See Piettre, S. R. and Baltzer, S., Tetrahedron Lett. (1997), 38, 1197-1200.
The preparation of 1,2-phenylenediboronic acid has been previously described on only one occasion (Clement, R., Thiais, F., Hebd. Seances Acad. Sci., Ser. C (1966), 263, 1398-400. The preparation of 1,2-phenylenediboronate complexes prepared from 2,2xe2x80x2-(1,2-phenylene)bis-[(4R,5R)-4,5-diphenyl-1,3,2-dioxaborolane], involving addition of either one or two equivalents of benzylamine, has been recently described. See Nozaki, K., Yoshida, M. and Takaya, H., Angew. Chem. Int. Ed. Engl. (1994), 33, 2452-2454 and Nozaki, K., Yoshida, M. and Takaya, H., Bull. Chem. Soc. Jpn. (1996), 69, 2043-2052.
2-Acetamidophenylboronic acids have been proposed as potential linkers for selective bioconjugation via the vicinal diol moieties of the carbohydrate residues associated with glycoproteins. See Cai, S. X. and Keana, J. F. W., Bioconjugate Chem. (1991), 2, 317-322. Phenylboronic acid bioconjugates derived from 3-isothiocyanatophenylboronic acid have been successfully utilized for appending radioactive technetium dioxime complexes to monoclonal antibodies for use in medical imaging. See Linder, K. E., Wen, M. D., Nowotnik, D. P., Malley, M. F., Gougoutas, J. Z., Nunn, A. D. and Eckelman, W. C., Bioconjugate Chem. (1991), 2, 160-170; Linder, K. E., Wen, M. D., Nowotnik, D. P., Ramalingam, K., Sharkey, R. M., Yost, F., Narra, R. K. and Eckelman, W. C., Bioconjugate Chem. (1991), 2, 407-414.
3-Aminophenylboronic acid has been covalently appended to proteins by a variety of chemical methods and the resulting phenylboronic acid bioconjugates tested for their binding of D-sorbitol, D-mannose and glycated hemoglobin (GHb). The interactions proved to be reversible and of very low affinity rendering the bioconjugates of very limited practical use. Similarly, an Alkaline Phosphatase-phenylboronate bioconjugate used in an attempted enzyme-linked assay for the detection of GHb failed to detect the presence of glycated protein. See Frantzen, F., Grimsrud, K., Heggli, D. and Sundrehagen, E., Journal of Chromatography B (1995), 670, 37-45.
In addition to attempts to utilize immobilized phenylboronates for chromatographic separation of biological molecules having the requisite functionalities, a novel class of phenylboronic acid reagents, phenyldiboronic acid reagents, and boronic and diboronic compound complexing reagents have been developed for conjugating biologically active species (or bioactive species) and exploiting indirect bioconjugation through reversible formation of a boronic acid complex. These reagents and associated conjugates may be used in a manner analogous to Avidin-Biotin and Digoxigenin-anti-Digoxigenin systems. However, unlike the Avidin-Biotin and Digoxigenin-anti-Digoxigenin systems where the viability of the biological macromolecule must be maintained to preserve requisite binding properties, the bioconjugate formed through the boron complex is generally insensitive to significant variations in ionic strength, temperature, the presence of organic solvents, and the presence of chaotropic agents (protein and nucleic acid denaturants). Phenylboronic acid reagents and boronic compound complexing reagents, their conjugates and bioconjugates as well as methods for their preparation and use are the subject of U.S. Pat. Nos. 5,594,111, 5,623,055, 5,668,258, 5,648,470, 5,594,151, 5,668,257, 5,688,928, 5,744,727, 5,777,148, 5,831,045, 5,831,046, 5,837,878, 5,847,192, 5,852,178, 5,859,210, 5,869,623, 5,872,224, 5,876,938, 5,877,297, and 6,008,406. 1,3- and 1,4-phenylene diboronic acid reagents and 1,3- and 1,4-diboronic compound complexing reagents, their conjugates and bioconjugates as well as methods for their preparation and use are the subject of U.S. Pat. Nos. 6,075,126, 6,124,471, and 6,156,884.
Phosphoramidite reagents containing protected 1,2-phenylenediboronic acid moieties for the preparation of modified synthetic oligonucleotides are the subject of U.S. Pat. No. 6,031,117. Oligonucleotides and polynucleotides containing 1,2-phenylenediboronic acid moieties, prepared from the aforementioned phosphoramidite reagents, are the subject of U.S. Pat. No. 6,013,783.
Notwithstanding the substantial amount of research into bioconjugation, and the substantial amount of investment in this field, the selectivity of 1,2-phenylenediboronic acid has not heretofore been successfully exploited to enable the conjugation of biological macromolecules (other than synthetic oligonucleotides and polynucleotides) with one another or with other molecular species that add useful properties.
The present invention relates to a novel class of 1,2-phenylenediboronic acid (1,2-PDBA) reagents useful for the preparation of bioconjugates, and the method of making and using such reagents. In one embodiment, the 1,2-PDBA reagents of the present invention are preferably complexed with boronic compound complexing reagents derived from salicylhydroxamic acid (SHA), or derivatives thereof. In a second embodiment, the 1,2-PDBA reagents of the present invention are preferably complexed with boronic compound complexing reagents derived from 2,6-dihydroxybenzohydroxamic acid (DHBHA), or derivatives thereof.
In the present invention, in the place of prior art Avidin-Biotin and Digoxigenin anti-Digoxigenin systems, 1,2-PDBA reagents are utilized in conjunction with boronic compound complexing reagents to facilitate chemical conjugation without the use of intermediary biological macromolecules. Bioconjugate preparation often involves the conjugation of several components including, but not limited to, proteins, peptides, polysaccharides, hormones, nucleic acids, liposomes and cells, with each other or with any other molecular species that add useful properties, including, but not limited to, drugs, radionuclides, toxins, haptens, inhibitors, fluorophores, ligands, and solid-phase supports including chromatographic supports. These various components utilized in bioconjugate preparation will collectively and individually be termed biologically active species or bioactive species.
Reagents suitable for the modification of a bioactive species for the purpose of incorporating one or more 1,2-phenylenediboronic acid moieties for subsequent conjugation to a different (or the same) bioactive species having one or more pendant boronic compound complexing moieties are of General Formula I, 
wherein group R is a reactive electrophilic or nucleophilic moiety suitable for reaction of the 1,2-phenylenediboronic acid reagent with a bioactive species. Group Z is a spacer and group Q is an optional linkage that includes one of amide, ether and thioether moieties.
Reagents of General Formula I exhibit superior properties when compared to prior art phenylboronic acid reagents, in that, they incorporate two equivalents of boronic acid per reactive group R, and afford complexes in conjunction with boronic compound complexing reagents that exhibit superior stability in aqueous systems.
Reaction of a reagent of General Formula I with a bioactive species affords a conjugate having pendant 1,2-phenylenediboronic acid moieties (one or more) of General Formula II, 
wherein the symbol labeled BAS represents a biologically active species (or bioactive species) that may or may not contain a portion of a reactive moiety (which may itself have a spacer) used to attach the bioactive species to the reagent. Group Z in General Formula II is a spacer and group Q is an optional linkage that includes one of amide, ether and thioether moieties. It will be appreciated that, in many embodiments, several identical reagents of the general formula of General Formula I will react with a single BAS molecule. For example, if the BAS is a protein, many 1,2-PDBA reagents will react with the protein, each reacting at one of the several sites on the protein which are reactive with the R group of the 1,2-PDBA reagent. A representative illustration of multiple 1,2-PDBA reagents reacted with a biologically active species (or bioactive species) includes the following: 
Conjugates of General Formula II exhibit reduced hydrophobic secondary properties as compared to phenylboronic acid conjugates known in the prior art. This reduction in hydrophobic secondary properties lowers the extent of nonspecific binding, which is known to be a problem associated with bioactive species that have been conjugated with several hydrophobic moieties.
To form an indirect bioconjugate without the use of an intermediary macromolecule the conjugate of General Formula II may be complexed with a boronic compound complexing conjugate. For example, boronic compound complexing reagents may be appended to a biologically active species to afford a conjugate having pendant boronic compound complexing moieties (one or more) of the general formula of General Formula III, 
wherein the symbol labeled BAS* represents a second bioactive species, that may include a linker portion and that may differ from the bioactive species labeled BAS. The BAS* may also include a portion of a reactive moiety used to attach the bioactive species to the boronic compound complexing reagent. Group X is an acidic electron pair donating species, and group Y is selected from one of O, S and NH.
A conjugate of General Formula II, with at least one biologically active species and having pendent 1,2-phenylenediboronic acid moieties (one or more), may be complexed with one or more conjugates of General Formula III, prepared from a second bioactive species BAS*, and having pendant boronic compound complexing moieties (one or more), to afford, for example, a bioconjugate of General Formula IV, 
wherein the symbols labeled BAS and BAS*, and groups Z and Q are as were previously defined. Group X is an acidic electron pair donating species, and group Y is selected from one of O, S and NH. In this manner, biological macromolecules may be conjugated to one another or with other functionalities that impart useful properties.
Alternatively, boronic compound complexing reagents may be appended to a biologically active species to afford a conjugate having pendant boronic compound complexing moieties (one or more) of General Formula V, 
wherein the symbol labeled BAS* represents a second bioactive species, that may include a linker portion and that may differ from the bioactive species labeled BAS. The BAS* may also include a portion of a reactive moiety used to attach the bioactive species to the boronic compound complexing reagent. Group X is an acidic electron pair donating species, and group Y is selected from one of O, S and NH.
A conjugate of General Formula II, with at least one biologically active species and having pendent 1,2-phenylenediboronic acid moieties (one or more), may be complexed with one or more conjugates of General Formula V, prepared from a second bioactive species BAS*, and having pendant boronic compound complexing moieties (one or more), to afford, for example, a bioconjugate of General Formula VI, 
wherein the symbols labeled BAS and BAS*, and groups Z and Q are as were previously defined. Group X is an acidic electron pair donating species, and group Y is selected from one of O, S and NH. In this manner, biological macromolecules may be conjugated to one another or with other functionalities that impart useful properties.
Bioconjugates of General Formulas IV and VI may be prepared in buffered aqueous solution or organic solvents. The bioconjugate is formed within a few minutes over a range of temperatures of from about 4xc2x0 C. to 70xc2x0 C. The stability of the bioconjugate in aqueous solution at a given pH and temperature is significantly influenced by groups X and Y.
The bioconjugation reaction (boronic acid complexation) is generally insensitive to significant variations in ionic strength, the presence of organic solvents, the presence of detergents, and the presence of chaotropic agents (protein denaturants), which are incompatible with prior art indirect labeling systems wherein the structure of a biological macromolecule must be maintained to preserve requisite binding properties. In most instances, the constraints governing the formation of bioconjugates, by the system herein described, are limited to those imposed by the conditions required to maintain viability (native conformation) of the bioactive species.